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Background and objective: Detection of genomic rearrangements like ALK fusions are mandatory in non-small cell lung cancer (NSCLC) as those alterations can be targeted by an increasing number of drugs. Fluorescence in-situ hybridization (FISH) is the gold standard but multiplexed technologies allow the analysis of several targets simultaneously. We have evaluated a novel RT-PCR based assay for the detection of ALK, ROS1 and RET rearrangement in NSCLC patients in this research study. Materials and Methods: FFPE tissue sections from 309 patients with late stage NSCLC were screened for ALK and ROS1 status using immunohistochemistry (IHC) and confirmed with FISH. Total nucleic acids were extracted from tissue sections using the Maxwell RSC RNA FFPE kit (Promega). Fusions were detected by RT-PCR using a prototype ALK/RET/ROS1 Fusion Panel assay (Roche). A subset of discordant cases were further analyzed by RNA sequencing using the QIAseq Targeted RNAscan Lung Cancer Panel (Qiagen). Results: All 309 samples were tested by RT-PCR with a 0% invalid rate. ALK fusions were detected in 39 samples: 29 were ALK FISH+, 5 were ALK FISH not contributive, 3 were ALK FISH- (less than 15% tumor cells presenting a gene fusion), and 2 were ALK IHC- and not tested by FISH. ROS1 fusions were detected in 12 samples: 9 were ROS1 FISH+, 1 was ROS1 FISH-, and 2 were ROS1 IHC- and not tested by FISH. RET fusions were detected in 3 samples. The remaining 255 samples were RT-PCR-: 5 were ALK FISH+ containing fusions not covered by the RT-PCR assay, 1 was ALK FISH+ containing ALK polysomy, 1 was ROS1 FISH+ containing a fusion not covered by the RT-PCR assay, 1 was ROS1 FISH+ containing ROS1 amplification, 1 was ROS1 FISH+ but ROS1 RNAseq-, and 246 samples were IHC- or IHC+/FISH-. The overall concordance rate between RT-PCR and FISH for ALK and ROS1 gene rearrangements was 91.9%. Although the RT-PCR assay did not detect 5 ALK and 1 ROS1 fusions due to design limitations, it did identify the presence of fusions in 9 tumors that were undetected by FISH (8 ALK and 1 ROS1). Conclusion: Using limited amount of biological material, RT-PCR was able to detect a substantial majority of ALK and ROS1 fusions identified by FISH, as well as fusions that were missed by the initial IHC/FISH screening. This study demonstrates RT-PCR as a feasible approach to detecting fusions in NSCLC. Citation Format: Marc G. Denis, Audrey Vallée, Christine Sagan, Guillaume Herbreteau, Sandrine Charpentier, Jaya Rajamani, Michael Lee, Ellen Ordinario. Detection of ALK, ROS1 and RET fusion transcripts in FFPE samples of non-small cell lung cancer patients using a novel RT-PCR based assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1271.

Background and objective: Detection of genomic rearrangements like ALK fusions are of great interest in non-small cell lung cancer (NSCLC) as those alterations can be targeted by an increasing number of drugs. To overcome tissue limitations, detection of these alterations from liquid biopsies is an unmet need, despite the development of novel NGS-based tests. To allow the detection of ALK rearrangements from circulating-free RNA (cfRNA) from NSCLC patients, we have evaluated a novel RT-PCR based assay and compared the results to tissue-based testing using immunohistochemistry (IHC) or fluorescence in-situ hybridization (FISH). Materials and Methods: Sixty-five patients with late stage NSCLC were included in the study. ALK status was assessed on tissue section using immunohistochemistry and/or FISH. cfRNA was extracted from 2 ml of plasma from EDTA or Streck BCT DNA tubes using a prototype cfRNA Sample Preparation method (Roche Molecular Systems, Pleasanton, CA). For the detection of ALK-rearrangements from plasma, a prototype ALK/RET/ROS1 Fusion Panel assay (Roche) was used. Results: The analytical sensitivity of the assay was evaluated by testing plasma from healthy individuals spiked with RNA extracted from FFPE tissue section of an ALK positive tumor. Positive results were obtained with samples spiked with as little as 3 ng RNA. Of the forty-five ALK-fusion positive patients, 11 patients were already under anti-ALK therapy and in none of the patients an ALK fusion could be detected. However, of five samples tested at progression, two (40%) were tested positive. For the 29 ALK-fusion positive samples which were included at baseline prior to therapy, 11 samples have been tested positive for ALK fusions (38%). All 20 negative controls were negative using the PCR based assay. Consequently, the key test parameters for samples tested at baseline were: sensitivity = 37.93% [95% CI 20.69% - 57.74%]; specificity = 100.00% [95% 83.16% - 100.00%]; positive predictive value = 100%; negative predictive value weighted for a prevalence of ALK fusions of 4% in metastatic NSCLC patients = 97.48% [95% CI 96.68 - 98.09]. Conclusion: The prototype cobas ALK/RET/ROS1 Fusion Panel assay was able to detect ALK fusion transcripts in the plasma of NSCLC patients at baseline as well as at disease progression. Limited sensitivity could be explained by biological factors influencing nucleic acid shedding by tumours, as well as the presence of fusions not covered by the assay. However, the assay demonstrated high specificity. These data demonstrate that this assay could potentially be used to select patients for an anti-ALK therapy when tissue samples are not available. Citation Format: Simon Heeke, Marius Ilié, Maryline Allegra, Audrey Vallée, Carole Salacroup, Virginie Tanga, Véronique Hofman, Jaya Rajamani, Michael Lee, Ellen Ordinario, Marc G. Denis, Paul Hofman. Detection of ALK fusion transcripts in plasma of non-small cell lung cancer patients using a novel RT-PCR based assay [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5299.